Much is know about the the proteins that function presynaptically in R7 growth cones to regulate their target selection, but essentially nothing is know about the postsynaptic requirement of R7-target selection. A complete understanding of the mechanisms of R7 target selection requires the identification of connectivity cues expressed by the R7-target neurons. To achieve that, we need to first identify the interneurons that serve as synaptic targets of R7axons. Second, we need a genetic method to manipulate these neurons. By screening Gal4 enhancer trap lines and subsequently analyzing these lines with the single-cell mosaic technique, we identified seven types of medulla interneuron neurons that likely receive input from one or two color channels. In particular, the small-field Tm5 type neurons extend dendritic arbors in the M2 (L2) and M3 (R8) layers and might convey both green and blue color information to the higher visual center lobula. Similarly, the small-field Tm20 type neurons connect both R7 (UV channel) and R8 (blue) inputs to a specific layer in the lobula. The Tm5 and Tm20 neurons might function as color-opposing neurons, which calculate intensity differences between different spectra. These findings suggest that color-vision circuits in insects and in primates share surprising similarity. Furthermore, these results highlight the lobula ganglion as the higher visual center for color vision. We are currently analyzing the connection patterns of these neurons at the EM level and testing how electrically silencing these neurons affects wavelength-selection behaviors. To genetically manipulate medulla target neurons, we need to express yeast recombinase (flipase) before they undergo the last cell division and commit to their final cell fates. First, we screened Gal4/lacZ enhancer trap lines from both public stock and our collections for expression in medulla precursor cells. We have successfully identified one such enhancer trap line, which expresses Gal4 in the medulla and lamina precursor cells. Second, we constructed a flipase-based enhancer trap vector, pP{flip}, and generated transgenic flies. Third, we used the P-element-swap technique to replace the exiting enhancer trap vector with our P{flip} vector. The resulting flies express flipase in the medulla and lamina precursor cells during development. Because the flip expression is under the direct control of the promoter identified in the original enhancer trap, it can be used in combination with the existing MARCM (mosaic analysis with a repressible cell marker). We demonstrated that this P{flip}/MARCM system is capable of generating and visualizing mosaic medulla neurons. We are currently testing a number of candidate genes for their requirement in medulla target neurons for R7 connectivity. In addition to directed mosaic analysis, the P{flip} enhancer trap system has two additional applications: lineage tracing and combinatorial gene expression. To trace the lineage of the medulla precursor cells, we combined this P{flip} system with a conditional reporter, Actin-Gal4 UAS-GFP. The flipase expressed in the medulla precursor cells removes the interruption cassette, thereby allowing the constitutive actin promoter to drive GFP expression in the progenitors of the medulla precursor cells even after the flipase expression has subsided. Using this lineage tracing system, we are able to trace the progenitors of the medulla precursor cells from the late larval to adult stages. For combinatorial gene expression, we combined the P{flip} system with a Gal4 enhancer trap and a conditional reporter, UASGFP. We were able to restrict the GFP (or any transgene) expression in the subset of neurons that expresses both flipase and Gal4. This combinatorial gene expression system has many potential uses for manipulating and analyzing small subsets of neurons for behavioral or histological studies.